Introduction to Enzymes

Thousands of biochemical reactions proceed at any

given instant within living cells. These reactions are
catalyzed by enzymes;
Enzymes are mostly proteins. But two important
enzymes are most certainly to be RNA (ribozymes).
One is the ribosome (peptidyl transfer) and the other
is the splicesome (splicing of intron);
Enzymes are the agents of metabolic function.
Enzymes play key functions in controlling rate of
reaction, coupling reactions, and sensing the
momentary metabolic needs of the cell.
Introduction to Enzyme Kinetics
Kinetics concerns with the rates of chemical reaction.
Enzyme kinetics addresses the biological roles of
enzymatic catalysts and quantify the remarkable
function of biological enzymes;
Enzyme kinetics information can be exploited to
control and manipulate the course of metabolic
events. Pharmaceuticals or drugs are often special
inhibitors targeted at a particular enzyme. Thus the
science of pharmacology relies on such information


CLASSES of enzymes: What they do:

Oxidoreductase transfers electrons
Transferase transfers molecules
Hydrolase breaks only O-H bonds
Lyase breaks bonds
Isomerase twists bonds
Ligase makes bonds

http://www.rcsb.org/pdb/
How to measure how good the enzyme is?




enzymatic units. Amount of enzyme needed to get a certain level
of activity (rate of reaction -moles product/unit time) under some
controlled conditions.

Originally developed because extractions are dirty, and hard to
guarantee all enzymes are functioning ones.

Genetically engineered enzymes can be more concentrated and
pure.
Get activity from initial slope of fig 1.2 (NEXT SLIDE)
Have to have substrate IN EXCESS to get activity


The Rate Law of Unimolecular reactions
Consider a reaction of overall stoichiometry,

The rate, or velocity, v of this reaction is the amount of P formed or
the amount of A consumed per unit time. Thus:


Rate law states that:


Where k is rate constant. v is a function of [A] to the first power, or
the first order. k is called first order constant.
dt
A d
v or
dt
P d
v
] [ ] [
= =
P A
] [
] [
A k
dt
A d
v = =
The Rate Law of Biomolecular Reactions
Consider a reaction of overall stoichiometry,

The rate, or velocity, v of this reaction is the amount of P or Q
formed or the amount of A or B consumed per unit time. Thus:



Rate law states that:


Where k is rate constant. v is a function of [A][B], or second order.
k is the second order rate constant.
dt
B d
dt
A d
v or
dt
Q d
dt
P d
v
] [ ] [ ] [ ] [
= = = =
Q P B A + +
] ][ [
] [
B A k
dt
A d
v = =
Rate Constant and Free Energy of
Activation


AG
(non
catalyzed)
AG
(catalyzed)
AG

P A X

Arrhenius Equation:


Enzymes do not alter the
potential of a chemical
reaction (i.e., Gibbs free
energy). They accelerate
the rate of reaction. In another
word, they lower the
activation energy.

Enzyme can accelerate
the rate of a reaction by as
much as 10
16
.
RT
G
Ae k
+
A

=
Why does an enzyme accelerate the rate?

ES =
EP
AG =

The BINDING equilibrium constant is
related to AG




AG is related to the relative increase in the
rate of a reaction
HOW?

( )
|
|
|
.
|

\
|
A
=
RT
o
G
S
B
K exp
BINDING
ACTIVATING
THE COMPLEX
ACTIVATING S BINDING TO
ACTIVATED S
How does the enzyme change AG?
AG = AH - TAS
1.) reduces enthalpy of transition state
2.) reduces entropy of transition

enzyme
R1
R2
entropy
mechanism

AKA
approximation
catalyst
stable state
activated state
H
o
enzyme
The amount of reduction depends on the strength of binding, which can be expressed in terms of the equilibrium
constant for binding K
B
.

In the most simplest case of MM model K
M
= K
B
= k
2


Experimental Measurements of Binding

Equilibrium dialysis:

A
T
= total amount of substrate
A
B
= total amount of BOUND substrate
A = total amount of UNBOUND substrate


| |
(

+ =
B
A A
T
A
(

= =
T
E
B
A
v occupied sites average . .#.

Detect physical change on binding
AA
Bi
= change in measured quantity
AA
BT
= total change in measured quantity

fraction of occupied sites =


|
.
|

\
|
A
A
= = O
BT
A
Bi
A
n
v
SINGLE SITE




K
B


P + A PA
| |
| || | A P
PA
Binding
K =
| || |
| | PA
A P
on Dissociati
K =
| |
| | | | PA P
PA
v
+
=
| || |
| | | || |
| |
| | A
B
K
A
B
K
A P
B
K P
A P
B
K
v
+
=
+
=
1

Multiple sites
Cooperative binding


Multiple sites
Cooperative binding

1 site
MULTIPLE SITES
P + A PA
PA + A PA
2
PA
2
+ A PA
3




ADAIR equation:



good for all types of binding


| || |
| || |
| |
| |

=
=
=
=
=
=
n
i
i
A
i
K
n
i
i
A
i
iK
n
i
i
A P
i
K
n
i
i
A P
i
iK
v
0
1
0
1
SPECIAL PLOTS


| |
( ) v n k
A
v
=
| |
| |

=
+
=
N
S
A
s
k
A
s
k
s
n
v
1
1
| |
| |

=
+
=
N
S
A
s
k
A
s
k
s
n
v
1
1
( )
(

v n
v
log
| | A log
plot

eqn

y-axis

x-axis

result

meaning

Scatchard

v

v/[A]

Straight
line

Non-cooperative
n equivalent sites
read n from y intercept

Scatchard


N kinds of sites

v

v/[A]

Curved
line

Non-cooperative
N inequivalent sites
read n
1
from y
intercept

Hill


N kinds of sites

sigmoid

Read k1 and k2 from 2
slopes; low[A] gives
strong sites, higher [A]
gives weaker sites
Cooperative/cooperati
ve

Enzyme Kinetics
The rate of unimolecular reaction is proportional to the concentration of
the reactant. Thus rate is linearily dependent on [A].




But if this reaction is catalyzed by an enzyme, the rate shows saturation
behavior. Why?


P A
] [
] [
A k
dt
A d
v = =
v
[A]
P A
Enzyme

v
[A]
The Mechaelis-Menten Equation
You need to know how this is derived


This is the complete chemical formula for an enzyme-catalyzed (E)
reaction of substrate, S and product, P;
Mechaelis-Menten equation describes the relationship between reaction
rate and substrate concentration. It can explain the saturation
behavior in catalyzed reactions as shown in the previous slide.
Mechaelis-Menten equation is derived based on the following three
conditions:
1) State steady assumption;
2) Initial velocity assumption;
3) Rate law.

P E ES S E + +
k
1
K
-1
k
2
K
-2
Steady State Assumption

Steady state is defined as the state during which the enzyme-
substrate complex, [ES], remains constant, or

Pre-steady state is the state during which [ES] builds up, usually
very fast;

MM equation concerns the reaction rate that is measured only
when the steady state has reached.
0
] [
=
dt
ES d
P E ES S E + +
k
1
k
2
K
-2
Initial Velocity Assumption


In the beginning of the reaction, there is very little product, or [P] is
small. So the amount of [ES] contributed by E+P is negligible.

Thus, the MM equation concerns the reaction rate that is measured
during early reaction period.
In which case, the enzyme catalyzed reaction can be modified to:

K
-1
P E ES S E + +
k
1
K
-1
k
2
P E ES S E + +
k
1
k
2
K
-2
Rate Law in Enzyme Catalyzed Reactions

Rate law still applies in enzyme catalyzed reactions.
The forward velocity, or rate, v
f
is,


The reverse velocity or rate, or the rate of disappearance v
d
is,


At steady state, there is no accumulation of [ES], thus:
P E ES S E + +
k
1
K
-1
k
2
| || | S E k v
f 1
=
| | | | | | ES k k ES k ES k v
d
) (
2 1 2 1
+ = + =

d f
v v =
Derivation of Michaelis-Menten Equation
We need one more condition, that is, the total enzyme concentration,
[E
t
] is the sum of that of enzyme-substrate complex, [ES], and that of
free enzyme, [E]:


At steady state, the forward rate should equal to the reverse rate:





Rate of production formation (rate law), v = k
2
[ES]. So:

| | | | | | | | | || | | || | | |
| | | || | | | | || | | | | |
| |
| || |
| |
| || |
| |
| |
| || |
| |
m
t k
k k
K
t t
t t
t t
K S
S E
ES
k
k k
S
S E
k k S k
S E k
ES
k k S k ES S E k ES k k S ES k E k
ES k k S ES k S E k ES k k S ES E k
m
+
=
+
+
=
+ +
=
+ + = + + =
+ = + =
+
=

1
2 1
) (
1
2 1
2 1 1
1
2 1 1 1 2 1 1 1
2 1 1 1 2 1 1
) (
)) ( (
)) ( ( ) (
) ( ) ( ) (
| | | | | | E ES E
t
+ = | || | | | | | | | S ES E k S E k v
t f
) (
1 1
= =
| || |
| |
m
t
K S
S E k
v
+
=
2
Notes on the MM Equations
The rate of production formation can usually be measured
experimentally by monitoring the progress curve of production
formation.
The maximum rate can be reached at saturating substrate
concentration, or when [S]


So MM equation can be re-written as:

| |
| |
m
K S
S v
v
+
=
max
| |
| |
max 2
] [
2
] [
1
v E k
E k
v
t
S
K
t
S
m
= =
+
=

Enzyme-catalyzed rate
is saturated
Understanding K
m
K
m
is a constant derived from rate constants

K
m
is, under true Michaelis-Menten conditions, an estimate of the
dissociation constant of E from S, because
at equilibrium,

Reversible reaction, dissociation constant is
So small K
m
means tight substrate binding; high K
m
means
weak substrate binding.
K
m
equals to the substrate concentration at which v=1/2v
max


1
2 1
k
k k
K
m
+
=

ES S E +
k
1
k
-1
| |
1
1
] [
] [
k
k
ES
S E
K
d

= =
| | ] [ ] [
1 1
ES k S E k

=
Understanding v
max
V
max
is asymptotically approached as substrate is increased, but
never reached in reality, because this requires that ALL enzymes
are bound with substrates (think about chemical equilibrium);




Turnover number, k
cat
, is defined as It describes
number of substrates converted into products per enzyme, per
unit time. When the enzyme is saturated with substrate, or
[S]>>[Et],

ES S E +
k
1
k
-1
] [
max
t
cat
E
v
k =
cat
t
k
E
v
k = =
] [
max
2
Orders of the MM Equation
(2
nd
, 1
st
, 0
th
orders)
When substrate concentration is low, it is first order in [S], or



Under the same low substrate concentration, [E
t
] [E], so it is
second order in [E] and [S] and k
cat
/K
m
is called apparent second-
order rate constant:


When substrate concentration is high, the MM equation is 0
th

order in [S]:

m
t cat
m
t cat
K
E k
K S
S E k
v
][S] [
] [
] ][ [
~
+
=
] [
] [
] ][ [
max
max
t
m
t
E v
K S
S E v
v ~
+
=
~
m
cat
m
t cat
K
E k
K
E k
v
][S] [ ][S] [
~ ~
The Catalytic Efficiency, k
cat
/K
m
k
cat
/K
m
is an estimate of "how perfect" the
enzyme is

The upper limit for k
cat
/K
m
is the diffusion limit
- the rate at which E and S diffuse together,
because:
m
cat
m
t cat
K
E k
K
E k
v
][S] [ ][S] [
~ ~
1
2 1
1 2 2
k
k k
k k
K
k
K
k
m m
cat
<
+
= ~

Things to know
List the two assumptions used to derive the
Michaelis-Menten equation;
Derive the Michaelis-Menten eqUation;
Define K
m
. State why K
m
describes the affinity of
enzyme for substrates;
Define K
cat
.
Plots of the MM Equation
(Be familiar with these plots)
The MM equation can be plotted as










Where K
m
corresponds to the horizontal axis when the vertical
axis is half way of V
max
value.

m
K S
S v
v
+
=
] [
] [
max
[S]
v
v
max

K
m

How to get K
M
and V
max
from real data?
Three kinds of plots:

Name

y-axis

x-axis

v
max


K
M


Lineweaver-
Burk

initial rate (mM/min)
-1


[S
0
]
-1


+

-

Eadie-Hofstee

initial rate (mM/min)

initial rate
(mM/min)
/[S
0
]
-1

+

+

Hanes

[S
0
]/initial
rate(mM/min)

[S
0
]

+

+


Lineweaver-Burk Plot of MM Equation
(Be familiar with these plots)

More conveniently, the MM equation can be plotted as a linear
plot by rearranging the expression above to:

] [
1 1
] [
] [ 1
] [
] [
max max max
max
S v
K
v S v
K S
v K S
S v
v
m m
m
- + =
+
=
+
=
1/[S]
1/v
1/v
max
-1/K
m

Slope=K
m
/v
max
Lineweaver-Burk, or double reciprocal Plot
Eadie-Hofstee Plot of MM Equation
(Be familiar with these plots)
More conveniently, the MM equation can be plotted as a linear plot
by rearranging the expression above to:

( )
] [
] [ ] [ ] [ ] [
] [
] [
max max max
max
S
v K
v v v K S v S v S v K S v
K S
S v
v
m
m m
m
= = = +
+
=
v/[S]
v
Slope=-K
m
Hanes-Wolff plot
(Be familiar with these plots)
More conveniently, the MM equation can be plotted as a linear
plot by rearranging the expression above to:

max max max
max
] [ ] [ ] [
] [
] [
v
K
v
S
v
K S
v
S
K S
S v
v
m m
m
+ =
+
=
+
=
[S]
[S]/v
K
m
/v
max
-K
m

Slope=1/v
max
Hanes-Wolff Plot
Enzyme Inhibition
Enzyme can be inhibited by inhibitors. Inhibitors are tools to scientists to
understand enzymes. Inhibitors are also in many cases pharmaceutical
reagents against diseases;

Inhibitors inhibit enzyme function by binding with enzymes. The binding
reaction can be either reversible or irreversible;

Reversible inhibitors associate with enzymes through non-covalent
interactions. Reversible inhibitors include three kinds:
Competitive inhibitors;
Non-competitive inhibitors;
Un-competitive inhibitors

Irreversible inhibitors associate with enzymes through covalent
interactions. Thus the consequences of irreversible inhibitors is to
decrease in the concentration of active enzymes.

Derivation of Michaelis-Menten Equation
We need one more condition, that is, the total enzyme concentration,
[E
t
] is the sum of that of enzyme-substrate complex, [ES], and that of
free enzyme, [E]:


At steady state, the forward rate should equal to the reverse rate:





Rate of production formation (rate law), v = k
2
[ES]. So:

| | | | | | | | | || | | || | | |
| | | || | | | | || | | | | |
| |
| || |
| |
| || |
| |
| |
| || |
| |
m
t k
k k
K
t t
t t
t t
K S
S E
ES
k
k k
S
S E
k k S k
S E k
ES
k k S k ES S E k ES k k S ES k E k
ES k k S ES k S E k ES k k S ES E k
m
+
=
+
+
=
+ +
=
+ + = + + =
+ = + =
+
=

1
2 1
) (
1
2 1
2 1 1
1
2 1 1 1 2 1 1 1
2 1 1 1 2 1 1
) (
)) ( (
)) ( ( ) (
) ( ) ( ) (
| | | | | | E ES E
t
+ = | || | | | | | | | S ES E k S E k v
t f
) (
1 1
= =
| || |
| |
m
t
K S
S E k
v
+
=
2
Competitive Reversible Inhibitors





)
] [
1 ( ] [
] [
max
I
m
K
I
K S
S v
v
+ +
=
v
[S]
v
max

K
m

1/[S]
1/v
1/v
max
-1/K
m

Slope=K
m
/v
max
P E ES S E + +
k
1
K
-1
k
2
EI
+
I
k
3
k
-3
K
m
increases
v
max
unchanged
+inhibitor
K
m
(1+[I]/K
I
)
-1/(K
m
(1+[I]/K
I
))
Competitive Reversible Inhibitors





)
] [
1 ( ] [
] [
max
I
m
K
I
K S
S v
v
+ +
=
v
[S]
v
max

K
m

1/[S]
1/v
1/v
max
-1/K
m

Slope=K
m
/v
max
P E ES S E + +
k
1
K
-1
k
2
EI
+
I
K
I
K
m
increases
v
max
unchanged
+inhibitor
K
m
(1+[I]/K
I
)
-1/(K
m
(1+[I]/K
I
))
Slope= K
m
(1+[I]/K
I
)/v
max
Noncompetitive (Pure) Reversible
Inhibitors





)
] [
1 (
] [
] [
max
I
m
K
I
v
K S
S
v
+
-
+
=
P E ES S E + +
k
1
k
-1
k
2
EI
+
I
K
I
K
m
unchanged
v
max
decreases
+
I
EIS
K
I
+inhibitor
1/[S]
1/v
1/v
max
-1/K
m

Slope=K
m
/v
max
(1+[I]/K
I
)/V
max
Slope= K
m
(1+[I]/K
I
)/v
max

v
[S]
v
max

K
m

K
m

V
max
/(1+[I]/K
I
)
+
S
Uncompetitive Reversible Inhibitors





)
] [
1 (
)
] [
1 (
] [
] [
max
I
K
v
I
K
K
S
S
v
I
I
m
+
-
+
+
=
P E ES S E + +
k
1
k
-1
k
2
K
m
decreases
v
max
decreases
Slope unchanged
+inhibitor
+
I
EIS
K
I
1/[S]
1/v
1/v
max
-1/K
m

Slope=K
m
/v
max
(1+ K
I
/[I])/V
max
Slope= K
m
/v
max

v
[S]
v
max

K
m
K
m
/(1+ K
I
/[I])
V
max
/(1+K
I
/[I])
- (1+ K
I
/[I])/K
m
Mixed Inhibitions
(Be familiar with these plots)






+inhibitor
1/[S]
1/v
1/v
max
-1/K
m

Slope=K
m
/v
max
+inhibitor
1/[S]
1/v
1/v
max
-1/K
m

Slope=K
m
/v
max
Summary of Classes of Inhibitors
Competitive inhibition - inhibitor (I) binds only to E, not
to ES
Noncompetitive inhibition - inhibitor (I) binds either to E
and/or to ES
Uncompetitive inhibition - inhibitor (I) binds only to ES,
not to E. This is a hypothetical case that has never
been documented for a real enzyme, but which makes
a useful contrast to competitive inhibition.
Mixed inhibition-when the dissociation constants of (I)
to E and ES are different. The inhibition is mixed.
Irreversible Inhibition
Irreversible inhibitors combine irreversibly with enzymes.

Several drugs in current medical use are irreversible inhibitors of
enzymes, or mechanism-based enzyme inactivators. For instance,
penicllin exerts its effects by covalently reacting with an essential
serine residue in the active site of glycoprotein peptidase, an
enzyme that acts to cross-link the peptidyglycan chains during
synthesis of bacterial cell walls.




Sample Questions
What is the v/V
max
ratio when [S]=5K
m
?





Draw a Lineweaver-Burk plot if V
max
=100 mol/mL, K
m
=2mM.



Draw the new Lineweaver-Burk plot on the same plot as above if
a competitive inhibitor is added. [I]=0.5 mM, K
I
=1mM.

6
5
5
5
] [
] [
] [
] [
max
max
=
+
=
+
=
+
=
m m
m
m m
K K
K
K S
S
v
v
K S
v S
v
Ribozymes
It was assumed that all enzymes are proteins until
1982 when Thomas Cech and Sydney Altman
discovered catalytic RNAs (Nobel, 1989 in Chemistry);
Catalytic RNA, or, ribozymes, satisfy several enzymatic
criteria: substrate specificity, enhance reaction rate,
and emerge from reaction unchanged;
Several know ribozymes:
RNase P: catalyzes cleavage of precursor tRNA molecules into
mature tRNAs;
Group I, II introns: catalyze their own splicing (cleaving and
ligating);
Ribosome: catalyzes peptidyl transfer reaction

Catalytic Antibodies: Abzymes
Antibodies are immunoglobulins. Antibodies are
elicited in an organism in response to immunological
challenge by a foreign molecule called antigens;
Antibodies elicited in response to transition state
analogs have the ability to stabilize the transition state
and thus can catalyze a reaction by forcing the
substrate into the transition state structure;